Life can survive environmental stress largely due to molecular chaperones. Beyond protein folding, chaperones represent a critical point of intervention in cancer, metabolic, and aging diseases, because these diseases are often dependent on proteins that require chaperones. Despite their ubiquitous importance, most chaperone mechanisms are poorly understood. The goal of this proposal is to elucidate the mechanism of a key chaperone in the endoplasmic reticulum, Grp94. Grp94 is a member of the Hsp90 family of molecular chaperones, which are essential in metazoans but whose mechanism is poorly understood. Grp94 is required for the folding of insulin-like growth factors (IGF1, IGF2) Insulin has a storied history, but large-scale folding of insulin is inefficient due to misfolding nd aggregation inherent to the entire IGF family. In contrast, just a few grams of pancreatic beta-cells can produce insulin for the lifetime of an individual. This discrepancy indicates mechanisms of IGF folding in the cell that are fundamentally different from in-vitro folding. Revealing the mechanism Grp94-mediated IGF folding requires understanding how and why IGFs misfold. The goal of Aim 1a is to define an experimentally determined folding and misfolding energy landscape for IGF2. To achieve this I will take advantage of a unique folding characteristic of the IGF family. Briefly, IGF refolding results in disulfide-trapped folding and misfolding intermediates. It has long been known that these intermediates can be separated chromatographically, and as a result, numerous states on the IGF folding energy landscape can be studied. The IGF folding and misfolding energy landscape will be used in Aim 1b to evaluate how Grp94 alters the process by which IGFs fold. Findings from this in-vitro approach can immediately be tested in-vivo with an already established cell model, as described in Aim 2. Together, these experiments will show how IGF proteins are folded efficiently in the cell with the aid of molecular chaperones. The large-scale folding of insulin is inefficient, which has an economic and environmental cost. One way insulin is produced in large-scale is via yeast secretion. However, for unknown reasons, native proinsulin is not secreted but insulin analogs with shortened C- peptides can be secreted. Since yeasts do not express Grp94, the goal of Aim 3 is to test whether Grp94 can enable yeast to secrete native proinsulin.